Mar Janna Dahl

Ibuprofen-Induced Patent Ductus Arteriosus Closure: Physiologic, Histologic, and Biochemical Effects on the Premature Lung

OBJECTIVE. The goal was to study the pulmonary, biochemical, and morphologic effects of a persistent patent ductus arteriosus in a preterm baboon model of bronchopulmonary dysplasia. METHODS. Preterm baboons (treated prenatally with glucocorticoids) were delivered at 125 days of gestation (term: 185 days), given surfactant, and ventilated for 14 days. Twenty-four hours after birth, newborns were randomly assigned to receive either ibuprofen (to close the patent ductus arteriosus; n = 8) or no drug (control; n = 13). RESULTS. After treatment was started, the ibuprofen group had significantly lower pulmonary/systemic flow ratio, higher systemic blood pressure, and lower left ventricular end diastolic diameter, compared with the control group. There were no differences in cardiac performance indices between the groups. Ventilation index and dynamic compliance were significantly improved with ibuprofen. The improved pulmonary mechanics in ibuprofen-treated newborns were not attributable to changes in levels of surfactant protein B, C, or D, saturated phoshatidylcholine, or surfactant inhibitory proteins. There were no differences in tracheal concentrations of cytokines commonly associated with the development of bronchopulmonary dysplasia. The groups had similar messenger RNA expression of genes that regulate inflammation and remodeling in the lung. Lungs from ibuprofen-treated newborns were significantly drier (lower wet/dry ratio) and expressed 2.5 times more epithelial sodium channel protein than did control lungs. By 14 days after delivery, control newborns had morphologic features of arrested alveolar development (decreased alveolar surface area and complexity), compared with age-matched fetuses. In contrast, there was no evidence of alveolar arrest in the ibuprofen-treated newborns. CONCLUSIONS. Ibuprofen-induced patent ductus arteriosus closure improved pulmonary mechanics, decreased total lung water, increased epithelial sodium channel expression, and decreased the detrimental effects of preterm birth on alveolarization.

Nasal Ventilation Alters Mesenchymal Cell Turnover and Improves Alveolarization in Preterm Lambs

RATIONALE Bronchopulmonary dysplasia (BPD) is a frequent cause of morbidity in preterm infants that is characterized by prolonged need for ventilatory support in an intensive care environment. BPD is characterized histopathologically by persistently thick, cellular distal airspace walls. In normally developing lungs, by comparison, remodeling of the immature parenchymal architecture is characterized by thinning of the future alveolar walls, a process predicated on cell loss through apoptosis. OBJECTIVES We hypothesized that minimizing lung injury, using high-frequency nasal ventilation to provide positive distending pressure with minimal assisted tidal volume displacement, would increase apoptosis and decrease proliferation among mesenchymal cells in the distal airspace walls compared with a conventional mode of support (intermittent mandatory ventilation). METHODS Accordingly, we compared two groups of preterm lambs: one group managed by high-frequency nasal ventilation and a second group managed by intermittent mandatory ventilation. Each group was maintained for 3 days. MEASUREMENTS AND MAIN RESULTS Oxygenation and ventilation targets were sustained with lower airway pressures and less supplemental oxygen in the high-frequency nasal ventilation group, in which alveolarization progressed. Thinning of the distal airspace walls was accompanied by more apoptosis, and less proliferation, among mesenchymal cells of the high-frequency nasal ventilation group, based on morphometric, protein abundance, and mRNA expression indices of apoptosis and proliferation. CONCLUSIONS Our study shows that high-frequency nasal ventilation preserves the balance between mesenchymal cell apoptosis and proliferation in the distal airspace walls, such that alveolarization progresses.

Vasculogenesis drives pulmonary vascular growth in the developing chick embryo.

Formation of the pulmonary vasculature has been described as occurring by outgrowth of existing vessels (angiogenesis), de novo formation of new vessels (vasculogenesis), or a combination of both processes. Uncertainty about the contribution of angiogenesis and vasculogenesis to pulmonary vascular formation is partly due to methodologic approaches. Evidence in favor of angiogenesis stems from studies that used vascular‐filling methods. Such methods identify only directly continuous lumina. Evidence for vasculogenesis has been provided by the use of molecular markers of blood vessel endothelium. Use of both methods has not been combined in the same species, however. We hypothesized, based on published evidence from quail and mouse, that chick pulmonary vascular formation occurs by vasculogenesis. To test that hypothesis, we used vascular filling, serial section, and immunohistochemical methods to analyze the developing lungs of chick embryos from Hamburger and Hamilton stages 20 to 43. Vascular filling suggested that the lumen of the pulmonary arteries sprouted from the sixth pharyngeal arch arteries. However, serial sections and immunohistochemical localization of fetal liver kinase‐1 protein, the receptor for vascular endothelial growth factor, showed that the pulmonary arterial tree formed from endothelial cell precursors and coalescence of isolated blood vessels in the mediastinal splanchnic mesenchyme centrally to the developing lung tissue distally. Pulmonary veins grew from the left atrium to the developing lungs. Pulmonary blood vessel formation occurred continuously throughout the embryonic period studied. Our results show that vasculogenesis is the main process by which the pulmonary vasculature forms in the developing chick embryo. Developmental Dynamics 233:145–153, 2005.

IUGR decreases elastin mRNA expression in the developing rat lung and alters elastin content and lung compliance in the mature rat lung

Complications of intrauterine growth restriction (IUGR) include increased pulmonary morbidities and impaired alveolar development. Normal alveolar development depends upon elastin expression and processing, as well as the formation and deposition of elastic fibers. This is true of the human and rat. In this study, we hypothesized that uteroplacental insufficiency (UPI)-induced IUGR decreases mRNA levels of elastin and genes required for elastin fiber synthesis and assembly, at birth (prealveolarization) and postnatal day 7 (midalveolarization) in the rat. We further hypothesized that this would be accompanied by reduced elastic fiber deposition and increased static compliance at postnatal day 21 (mature lung). We used a well characterized rat model of IUGR to test these hypotheses. IUGR decreases mRNA transcript levels of genes essential for elastic fiber formation, including elastin, at birth and day 7. In the day 21 lung, IUGR decreases elastic fiber deposition and increases static lung compliance. We conclude that IUGR decreases mRNA transcript levels of elastic fiber synthesis genes, before and during alveolarization leading to a reduced elastic fiber density and increased static lung compliance in the mature lung. We speculate that the mechanism by which IUGR predisposes to pulmonary disease may be via decreased lung elastic fiber deposition.

Mechanism of Reduced Lung Injury by High-Frequency Nasal Ventilation in a Preterm Lamb Model of Neonatal Chronic Lung Disease

The mechanism underlying the potentially beneficial effects of the “gentler” modes of ventilation on chronic lung disease (CLD) of the premature infant is not known. We have previously demonstrated that alveolar parathyroid hormone-related protein-peroxisome proliferator-activated receptorγ (PTHrP-PPARγ) signaling is critically important in alveolar formation, and this signaling pathway is disrupted in hyperoxia- and/or volutrauma-induced neonatal rat lung injury. Whether the same paradigm is also applicable to CLD, resulting from prolonged intermittent mandatory ventilation (IMV), and whether differential effects of the mode of ventilation on the PTHrP-PPARγ signaling pathway explain the potential benefits of the “gentler” modes of ventilation are not known. Using a well-established preterm lamb model of neonatal CLD, we tested the hypothesis that ventilatory support using high-frequency nasal ventilation (HFNV) promotes alveolar PTHrP-PPARγ signaling, whereas IMV inhibits it. Preterm lambs managed by HFNV or IMV for 21 d following preterm delivery at 132-d gestation were studied by Western hybridization and immunofluorescence labeling for key markers of alveolar homeostasis and injury/repair. In lambs managed by IMV, the abundance of key homeostatic alveolar epithelial-mesenchymal markers was reduced, whereas it was significantly increased in the HFNV group, providing a potential molecular mechanism by which “gentler” modes of ventilation reduce neonatal CLD.

High-frequency nasal ventilation for 21 d maintains gas exchange with lower respiratory pressures and promotes alveolarization in preterm lambs

Background:Short-term high-frequency nasal ventilation (HFNV) of preterm neonates provides acceptable gas exchange compared to endotracheal intubation and intermittent mandatory ventilation (IMV). Whether long-term HFNV will provide acceptable gas exchange is unknown. We hypothesized that HFNV for up to 21 d would lead to acceptable gas exchange at lower inspired oxygen (O2) levels and airway pressures compared to intubation and IMV.Methods:Preterm lambs were exposed to antenatal steroids and treated with perinatal surfactant and postnatal caffeine. Lambs were intubated and resuscitated by IMV. At ~3 h of age, half of the lambs were switched to noninvasive HFNV. Support was for 3 or 21 d. By design, Pao2 and Paco2 were not different between groups.Results:At 3 d (n = 5) and 21 d (n = 4) of HFNV, fractional inspired O2 (FiO2), peak inspiratory pressure (PIP), mean airway, intratracheal, and positive end-expiratory pressures, oxygenation index, and alveolar–arterial gradient were significantly lower than matched periods of intubation and IMV. Pao2/FiO2 ratio was significantly higher at 3 and 21 d of HFNV compared to matched intubation and IMV. HFNV led to better alveolarization at 3 and 21 d.Conclusion:Long-term HFNV provides acceptable gas exchange at lower inspired O2 levels and respiratory pressures compared to intubation and IMV.

Pharmacokinetics of Budesonide Administered with Surfactant in Premature Lambs: Implications for Neonatal Clinical Trials.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature human infants, which may persist through adulthood. Airway inflammation has been firmly established in the pathogenesis of BPD. Previous studies to reduce airway inflammation with high-dose dexamethasone demonstrated adverse neurological outcomes, despite lower incidences of BPD. Instillation of budesonide and surfactant can facilitate early extubation and reduce the incidence of BPD and death among very low birth weight infants. However, the pharmacokinetics of budesonide and its distribution into the lung and brain are unknown. Therefore, 5 premature lambs were administered 0.25 mg/kg budesonide, with surfactant as the vehicle. Plasma and tissue samples were taken from the lambs for measurement of budesonide, 16α- hydroxy prednisolone, and budesonide palmitate using LC/MS/MS. Peak plasma budesonide concentrations were inversely correlated with the oxygenation index (correlation coefficient of -0.75). plasma budesonide concentrations were extremely low (~10% of expected) for two lambs that had high oxygenation indices and were excluded from further analyses. For the remaining 5 premature lambs, a non-compartmental analysis demonstrated an AUCinf of 148.77 ± 28.16 h*μg/L, half-life of 4.76 ± 1.79 h, and Cmax of 46.17 ± 17.71 µg/L. Using population pharmacokinetic methods, a onecompartment model with exponential residual error and first-order absorption adequately described the data. The apparent clearance and apparent volume of distribution of budesonide were estimated at 6.29 L/h (1.99 L/h/kg) and 29.1 L (9.2 L/kg), respectively. Budesonide and budesonide palmitate, but not 16α-hydroxy prednisolone, were detected in lung tissue. In this study, budesonide and its metabolites were not detected in the brain, which suggests that intratracheal instillation suggests that after local pulmonary deposition, there is no evidence of budesonide accumulation in the central nervous system. Overall, these results show that peak plasma budesonide concentrations are inversely correlated with the oxygenation index and that lung-specific delivery of budesonide avoids accumulation of budesonide in the brain.

Development of Mechanical and Failure Properties in Sheep Cerebral Arteries

Traumatic brain injury (TBI) is a devastating problem for people of all ages, but the nature of the response to such injury is often different in children than in adults. Cerebral vessel damage and dysfunction are common following TBI, but age-dependent, large-deformation vessel response has not been characterized. Our objective was to investigate the mechanical properties of cerebral arteries as a function of development. Sheep middle cerebral arteries from four age groups (fetal, newborn, juvenile, and adult) were subjected to biaxial loading around physiological conditions and then to failure in the axial direction. Results show little difference among age groups under physiological loading conditions, but response varied significantly with age in response to large axial deformation. Vessels from all age groups reached the same ultimate stretch level, but the amount of stress carried at a given level of stretch increased significantly with age through the developmental period (fetal to juvenile). Our results are the first to identify changes in cerebral vessel response to large deformations with age and may lead to new insights regarding differences in response to TBI with age.

Alveolar formation is dysregulated by restricted nutrition but not excess sedation in preterm lambs managed by noninvasive support

Background:Preterm birth and respiratory support with invasive mechanical ventilation frequently leads to bronchopulmonary dysplasia (BPD). A hallmark feature of BPD is alveolar simplification. For our preterm lamb model of BPD, invasive mechanical ventilation is associated with postnatal feeding intolerance (reduced nutrition) and sedation. In contrast, preterm lambs managed by noninvasive support (NIS) have normal alveolar formation, appropriate postnatal nutrition, and require little sedation. We used the latter, positive-outcome group to discriminate the contribution of reduced nutrition vs. sedation on alveolar simplification. We hypothesized that, restricted nutrition, but not sedation with pentobarbital, contributes to impaired indices of alveolar formation in preterm lambs managed by NIS.Methods:Preterm lambs managed by NIS for 21d were randomized into three groups: NIS control, NIS plus restricted nutrition, and NIS plus excess sedation with pentobarbital. We quantified morphological and biochemical indices of alveolar formation, as well as mesenchymal cell apoptosis and proliferation.Results:Restricted nutrition impaired morphological and biochemical indices of alveolar formation, and reduced mesenchymal cell apoptosis and proliferation. Excess sedation with pentobarbital did not alter these indices, although mesenchymal cell apoptosis was less.Conclusion:Our results demonstrate that restricted nutrition, but not excess sedation, contributes to impaired alveolar formation during the evolution of BPD in chronically ventilated preterm lambs.

Diffusion tensor imaging and histology of developing hearts

Diffusion tensor imaging (DTI) has emerged as a promising method for noninvasive quantification of myocardial microstructure. However, the origin and behavior of DTI measurements during myocardial normal development and remodeling remain poorly understood. In this work, conventional and bicompartmental DTI in addition to three‐dimensional histological correlation were performed in a sheep model of myocardial development from third trimester to postnatal 5 months of age. Comparing the earliest time points in the third trimester with the postnatal 5 month group, the scalar transverse diffusivities preferentially increased in both left ventricle (LV) and right ventricle (RV): secondary eigenvalues D2 increased by 54% (LV) and 36% (RV), whereas tertiary eigenvalues D3 increased by 85% (LV) and 67% (RV). The longitudinal diffusivity D1 changes were small, which led to a decrease in fractional anisotropy by 41% (LV) and 33% (RV) in 5 month versus fetal hearts. Histological analysis suggested that myocardial development is associated with hyperplasia in the early stages of the third trimester followed by myocyte growth in the later stages up to 5 months of age (increased average myocyte width by 198%, myocyte length by 128%, and decreased nucleus density by 70% between preterm and postnatal 5 month hearts.) In a few histological samples (N = 6), correlations were observed between DTI longitudinal diffusivity and myocyte length (r = 0.86, P < 0.05), and transverse diffusivity and myocyte width (r = 0.96, P < 0.01). Linear regression analysis showed that transverse diffusivities are more affected by changes in myocyte size and nucleus density changes than longitudinal diffusivities, which is consistent with predictions of classical models of diffusion in porous media. Furthermore, primary and secondary DTI eigenvectors during development changed significantly. Collectively, the findings demonstrate a role for DTI to monitor and quantify myocardial development, and potentially cardiac disease. Copyright